troubleshooting and risks in ngs detection of small subclones · 2019-12-12 · troubleshooting and...
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Troubleshooting and "risks" in NGS detection of small subclones
Panagiotis Baliakas, MD-PhD
Dept of Immunology, Genetics and Pathology Science for Life Laboratory, Uppsala University
Medical Genetics, Akademiska Hospital, Uppsala
NGS: an obligation or a choice?
Döhner H et al. Blood 2017
NGS: a dream or a nightmare?
• Non-uniform coverage due to bias
• Inefficient enrichment and sequencing of GC-rich regions
• Inaccurate quantification due to duplicates
• Low efficacy regarding large deletions/insertions
• Artifacts limit quantification and variant calling accuracy
Unique molecular identifiers (UMI): A promising approach
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1.000
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N10 N6 N11 N9 N5 N35N16N14N34 N8 N36N38N13 N7 N12N33 N3 N21N30N15N19 N4 N37 N1 N24N23N29N31N20N25 N2 N17N22N28N27N26N32N18
Ab
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Sample
Mean Standard Coverage Mean UMI Coverage
R. Rosenquist, LA. Sutton
Are there any other alternatives?
Lode L et al. Haematologica 2017
Singlemolecule real-time (SMRT) sequencing- Pacific Bioscienses (PacBio) platform
• supported ≥ 30 reads • VAF: 1% - 10% and found by at least one methodology
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T4 p.R248W T20 p.R273H T20 p.C135F T30 p.Y220C T41 p.R181H T44 p.G266R T44 p.N131I T48 p.R249K
VA
F %
TP53 gene mutations
HaloPlex center 1 HaloPlex center 2 Multiplicom center 1
Multiplicom center 2 TruSeq center 1 TruSeq center 2
R. Rosenquist, LA. Sutton
How can we define ”small” in the clinical setting?
Rossi D et al. Blood 2014
How can we define ”small” in the clinical setting?
Nadeu F et al. Blood 2016
ΤΡ53 mutations: Do all small clones matter?
Malcikova J. et al. Human Mutation 2014
Implementing NGS in the clinical setting
Is the patient mutated?
Should we report what we see or should we choose to not report what may be there?
Implementing NGS in the clinical setting
Clinical Genetics, Uppsala
Validation period Close collaboration with the clinicians
Number of pathogenic variants
n=32
TP53 mutated patients n=18 (12%)
7 patients with multiple
mutations
3 mutations with VAF 10-12%
All patients
n=146
Implementing NGS in the clinical setting
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7/32 (22%) mutations with VAF 5-10% 2/7 in patients with multiple mutations
Mean depth: 8039 reads
Clinical Genetics, Uppsala
Patient 1
Clinical Genetics, Uppsala
FISH (2017-03-23)
Biallelic interstitial deletion of 13q14.3 (78%)
NGS (2017-03-23)
Variant (Protein) VAF Depth Interpretation
p.Q38fs*6 10% 6903 Pathogenic
p.P152L 20% 10956 Pathogenic
p.R196* 11% 6647 Pathogenic
p.Y234C 21% 19844 Pathogenic
p.T284fs*21 9.8% 6574 Pathogenic
2 of the mutations were detected 2 years before with Sanger
Patient 2
Clinical Genetics, Uppsala
FISH (2017-08-23)
Monoallelic interstitial deletion of 13q14.3 (12%)
NGS (2017-08-23)
Variant (Protein) VAF Depth Interpretation
p.S215G 6% 3363 Pathogenic
VAF is lower than 10% but the variants is reported because: (1) it has been reported as pathogenic in various forms of cancer, and ; (2) of the high coverage which decreases the posibility of being a technical artifact. Re-analysis of a second sample showed the same variant with VAF of 7%.
Patient 3
Clinical Genetics, Uppsala
FISH (2017-08-23)
del(11q) (62%)
Biallelic interstitial deletion of 13q14.3 (12%)
NGS (2017-08-23)
Variant (Protein) VAF Depth Interpretation
p.R273C 5% 7444 Pathogenic
VAF is lower than 10% but the variants is reported because: (1) it has been reported as pathogenic in various forms of cancer, and ; (2) of the high coverage which decreases the posibility of being a technical artifact. In order to evaluate the actual clinical relevance of the reported variant we recommend a new analys in a new sample.
Patient 4
Clinical Genetics, Uppsala
FISH (2017-06-13)
No result
NGS (2017-06-13)
Variant (Protein) VAF Depth Interpretation
p.C275Y 8% 7671 Pathogenic
In order to evaluate the clinical significance of the variant a new analysis in a new sample is recommended.
FISH (2017-08-23)
del(17p) (73%)
NGS in the clinical setting
• A new companion that changed the routine practice
• Not perfect, but highly applicable
• Imperative to understand the limitations
• Collaborate with each other
• Collaborate and train clinicians
• Validate results in the context of clinical trials
Uppsala University Sujata Bhoi Diego Cortese Karin Larsson Viktor Ljungström Mattias Mattsson Larry Mansouri Lesley-Ann Sutton Emma Young Richard Rosenquist
CERTH, Thessaloniki Aliki Xochelli Evangelia Stalika Kostas Stamatopoulos
G.Papanicolaou Hospital Niki Stavroyianni Anastasia Athanasiadou Michalis Iskas Anna Vardi Vicky Douka Tasoula Touloumenidou Achilles Anagnostopoulos
IRCCS San Raffaele, Milan Lydia Scarfo Paolo Ghia
CEITEC, Brno Karla Plevova Jitka Malcikova Sarka Pospisilova
Hopital Pitie-Salpetriere, Paris Frederic Davi Florence Nguyen Khac
Royal Bournemouth Hospital David Oscier Zadie Davis
University of Eastern Piedmont Novara Davide Rossi Gianluca Gaidano
University of Southampton Jonathan C. Strefford
Spanish Cytogenetic Group Anna Puiggros Julio Delgado Pau Abrisqueta Rosa Collado M Jose Calasanz Neus Ruiz-Xiville Carolina Moreno Blanca Espinet
MLL Munich Leukemia Laboratory, Germany Sabine Jeromin Claudia Haferlach
Academic Medical Center Amsterdam Arnon Katter Alexander Leeksma
Clinical Genetics, Akademiska University Hospital Uppsala, Sweden
Lucia Cavelier
Monica Hermanson
Tatjana Pandzic
Marie Engvall
Kristin Ayoola
Gustafsson
Anna Bremer
